Article Text

Original research
Molecular mechanisms underlying the modulation of T-cell proliferation and cytotoxicity by immobilized CCL21 and ICAM1
  1. Sofi Yado1,
  2. Bareket Dassa2,
  3. Rawan Zoabi1,
  4. Shlomit Reich-Zeliger3,
  5. Nir Friedman4 and
  6. Benjamin Geiger1
  1. 1 Department of Immunology and Regenerative Biology, Weizmann Institute of Science, Rehovot, Israel
  2. 2 Bioinformatics Unit, Department of Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot, Israel
  3. 3 Department of Systems Immunology, Weizmann Institute of Science, Rehovot, Israel
  4. 4 Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
  1. Correspondence to Professor Benjamin Geiger; benny.geiger{at}weizmann.ac.il

Abstract

Background Adoptive cancer immunotherapy, using engineered T-cells, expressing chimeric antigen receptor or autologous tumor infiltrating lymphocytes became, in recent years, a major therapeutic approach for diverse types of cancer. However, despite the transformative potential of adoptive cancer immunotherapy, this field still faces major challenges, manifested by the apparent decline of the cytotoxic capacity of effector CD8+ T cells upon their expansion. To address these challenges, we have developed an ex vivo “synthetic immune niche” (SIN), composed of immobilized CCL21 and ICAM1, which synergistically induce an efficient expansion of antigen-specific CD8+ T cells while retaining, and even enhancing their cytotoxic potency.

Methods To explore the molecular mechanisms through which a CCL21+ICAM1-based SIN modulates the interplay between the proliferation and cytotoxic potency of antigen-activated and CD3/CD28-activated effector CD8+ T cells, we performed integrated analysis of specific differentiation markers via flow cytometry, together with gene expression profiling.

Results On day 3, the transcriptomic effect induced by the SIN was largely similar for both dendritic cell (DC)/ovalbumin (OVA)-activated and anti-CD3/CD28-activated cells. Cell proliferation increased and the cells exhibited high killing capacity. On day 4 and on, the proliferation/cytotoxicity phenotypes became radically “activation-specific”; The DC/OVA-activated cells lost their cytotoxic activity, which, in turn, was rescued by the SIN treatment. On longer incubation, the cytotoxic activity further declined, and on day7, could not be rescued by the SIN. SIN stimulation following activation with anti-CD3/CD28 beads induced a major increase in the proliferative phenotype while transiently suppressing their cytotoxicity for 2–3 days and fully regaining their killing activity on day 7. Potential molecular regulatory pathways of the SIN effects were identified, based on transcriptomic and multispectral imaging profiling.

Conclusions These data indicate that cell proliferation and cytotoxicity are negatively correlated, and the interplay between them is differentially regulated by the mode of initial activation. The SIN stimulation greatly enhances the cell expansion, following both activation modes, while displaying high survival and cytotoxic potency at specific time points following stimulation, suggesting that it could effectively reinforce adoptive cancer immunotherapy.

  • T cell
  • Adoptive cell therapy - ACT
  • Gene expression profiling - GEP
  • Immunotherapy
  • Tumor microenvironment - TME

Data availability statement

Data are available in a public, open access repository. The datasets supporting the conclusions of this article are included within the article and have been deposited in NCBI’s Gene Expression Omnibus (GEO) and is available through GEO series accession number GSE254335 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE254335). For further information on original data, contact the corresponding author.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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WHAT IS ALREADY KNOWN ON THIS TOPIC

  • Adoptive cancer immunotherapy, has become, in recent years, a major therapeutic approach for diverse types of cancer. However, despite its transformative potential, this field still faces major challenges, manifested by the apparent decline of the cytotoxic capacity of effector CD8+T cells on their expansion. To address these challenges, we have developed an ex vivo “synthetic immune niche” (SIN), composed of immobilized CCL21 and ICAM1, which synergistically induce an efficient expansion of antigen-specific CD8+ T cells while retaining, and even enhancing their cytotoxic potency.

WHAT THIS STUDY ADDS

  • The data indicate that cell proliferation and cytotoxicity are negatively correlated, and the interplay between them is differentially regulated by the mode of initial activation. The SIN stimulation greatly enhances the cell expansion, following both activation modes, while displaying high survival and cytotoxic potency at specific time points following stimulation. Transcriptional and multispectral imaging profiling highlighted potential molecular regulators of T-cell proliferation, cytotoxicity and exhaustion, which might participate in the SIN-mediated response.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

  • The results presented in this paper provide novel insights into the molecular mechanisms underlying the SIN effect in different therapy-related activation contexts, opening new approaches for enhancing the efficacy of adoptive cancer immunotherapy.

Background

Adoptive cancer immunotherapy involving the transfer of engineered T cells expressing chimeric antigen receptor (CAR) or autologous tumor-infiltrating lymphocytes (TILs) into patients with cancer is a highly promising approach for inducing an effective antitumor immune response.1–4 However, despite their remarkable clinical long-term therapeutic potential, these therapies still encounter major challenges. These include high variability in the tumor-specific response to therapy, as well as intrinsic differences in the properties of effector immune cells, their numbers and therapeutic potency.1 3 4 Thus, TILs are commonly applied for the treatment of solid tumors, such as metastatic melanoma and breast, ovarian, urothelial, endometrial and digestive tract cancers, but their success rates are still rather limited.5 6 CAR-T cells, on the other hand, are commonly used for the treatment of hematological malignancies, such as B-cell acute lymphoblastic leukemia and non-Hodgkin’s lymphoma.7 8 The limited success rates of both CAR-T-cell-based and TIL-based therapies are attributed to several factors, including intrinsic heterogeneity in the responsiveness of the target tumors, which is still poorly understood at the mechanistic and cellular levels6 8; immune suppression by the checkpoint system8; and, often, limited numbers of autologous T cells that are available for effective therapy following ex vivo expansion.5 6 Translating these requirements into numbers indicates that for launching a successful CAR-T-cell-based or TIL-based therapy, the number of potent T cells needed is on the order of 1×1010–1×1011 cells,9 10 which requires a major ex vivo expansion step on the order of ~1000- fold or greater. Unfortunately, at this level of expansion, effector T cells often exhibit reduced intrinsic cytotoxic potency, which is commonly attributed to exhaustion and anergy,11–13 as well as to elevated susceptibility to checkpoint suppression.14 Notably, while the complex interplay between T-cell proliferation, differentiation, dysfunction and exhaustion is widely recognized, the molecular mechanisms underlying these interactions are largely unknown.15–21

To address these challenges, our group and others have attempted in recent years to design novel expansion protocols that optimize both cell yield and potency; these protocols include the use of soluble or immobilized cytokines (eg, IL-2, IFN-γ, IL-6, and IL-107 22 23), chemokines and adhesion ligands (eg, CCL21 (chemokine C-C motif ligand 21) and ICAM1 (intercellular adhesion molecule 124 25). Specifically, we recently designed and characterized a 2D molecular scaffold that acts as a “synthetic immune niche” (SIN) that supports the ex vivo expansion of T cells while maintaining and even enhancing their intrinsic cytotoxic activity. CCL21 is a homeostatic chemokine that is produced by endothelial and stromal cells within the lymph node26 and plays key roles in different features of immune responses, including recruitment of T cells and dendritic cells (DCs) to lymphoid organs,27 28 enhancement of immune cell migration,29 priming of T cells for immune synapse formation,24 and stimulation of both naïve T-cell expansion and Th1 cell polarization.25 30 31 ICAM1 is a cell-cell adhesion molecule that participates in the formation of immune synapses and the promotion of T-cell activation through binding to its integrin receptor, LFA1 (lymphocyte function-associated antigen 1).32 These two components were previously shown to act synergistically; CCL21 increases LFA1 responsiveness to ICAM1 and mediates the arrest of motile lymphocytes on ICAM1-expressing DCs, endothelial cells, and other T cells within their microenvironment.33 34

In a previous study, we showed that incubating activated murine CD4+ 35 or CD8+ 15 T cells with an immobilized CCL21+ICAM-1 SIN augmented the expansion of both T-cell populations. Furthermore, ovalbumin (OVA)-specific CD8+ T cells cultured on this SIN displayed augmented cytotoxic activity toward OVA-expressing B16 melanoma target cells and effectively suppressed cancer development in mice inoculated with OVA-expressing B16 melanoma cells.15 Furthermore, recent studies have indicated that the CCL21+ICAM1 SIN affects patient-derived TILs and increases their expansion rate as well as the expression of specific activation markers in treated cells.36 Taken together, these findings provide compelling evidence for the capacity of CCL21+ICAM1 SIN to augment both the proliferation and killing capacity of CD8+ 15 T cells, two features that commonly display a negative correlation.36 However, the molecular and cellular mechanisms underlying the effects of this SIN on the cross-talk between T-cell expansion and functionality following different modes of activation are still unclear.

In the present study, we explored the differential temporal effects of SIN treatment on T-cell expansion, cytotoxicity and the expression of relevant molecular regulators and markers in CD8+ T cells activated either by DC/OVA or by anti-CD3/CD28-coated beads. We showed here that on day 3, following activation, the transcriptomic effect of SIN was similar for both activation routes; cell proliferation increased, and the cells showed high killing capacity. On day 4, on the other hand, the interplay between proliferation and cytotoxicity becomes radically different in the absence or presence of SIN in a highly ‘activation mode-specific’ manner. Thus, in the absence of SIN, the cytotoxic activity of DC/OVA-activated cells decreased, while the cells remained cytotoxic following SIN treatment. After longer incubation, the cytotoxic activity further decreased, and on day 7, it could not be rescued by SIN. Unlike these cells, CD3/CD28-activated cells, without SIN treatment, remained cytotoxic for 4–7 days but displayed low expansion rates. On SIN stimulation, the bead-activated cells became highly proliferative; however, their cytotoxic capacity was markedly suppressed, and their killing activity was restored only on day 7. These results indicate that cell proliferation and cytotoxicity are negatively correlated and that the interplay between them is differentially regulated by SIN in an activation mode-specific manner. Transcriptional and multispectral imaging profiling highlighted potential molecular regulators of T-cell proliferation, cytotoxicity and exhaustion, which might participate in the SIN-mediated response.

Materials and methods

Animals

C57BL/6 mice were obtained from Envigo Laboratories (Rehovot, Israel). T-cell receptor (TCR)-transgenic OT-I mice (harboring OVA-specific CD8+ T cells) were purchased from Jackson Laboratories (Bar Harbor, Maine, USA) and bred at the Department of Veterinary Resources of Weizmann Institute. For all the experiments, female mice aged between 6 and 12 weeks were used. Experiments were performed under protocols approved by the Weizmann Institute’s Institutional Animal Care and Use Committee.

CD8+ T-cell isolation, activation and culture

Naïve CD8+ T cells were purified (>95%) from a cell suspension and harvested from the crushed spleens of OT-I mice using a CD8a+ T-Cell Isolation Kit and magnetic-associated cell sorting (MACS) according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Similarly, DCs were purified (>90%) from the spleens of C57BL/6 mice using MACS CD11c microbeads (Miltenyi Biotec). Freshly isolated CD8+ T cells were then activated either with anti-CD3 and anti-CD28 antibody-coated microbeads at a 1:1 bead:cell ratio (Miltenyi Biotec) and with 100 U/mL IL-2 (BioLegend, San Diego, California, USA) (referred to as “Bead activated T cells” or “non-specific activation”) or by using OVA peptide (OVA257-264, InvivoGen, San Diego, California, USA; 1 µg/mL), which was loaded on DCs (used at a 3:1 T-to-DC ratio), referred to as “DC-activated T cells” or “antigen-specific activation”. T cells were suspended in RPMI 1640 medium without phenol red, supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, and 50 mM β-mercaptoethanol (Biological Industries, Beit Haemek, Israel) (“ complete medium”). The cells were further cultured at 37°C in a 96-well plate (250 µL/well) that was either uncoated or precoated with CCL21-ICAM1 SIN. On day 3 poststimulation, the beads were removed using a magnet, and the contents of all the wells were diluted two fold and subsequently distributed into new wells (coated and uncoated) with fresh medium and IL-2. DC-activated cells were similarly treated without the addition of IL2. The number of viable T cells was determined at selected time points after stimulation (3, 4, 6, and 7 days) using a CellDrop FL cell counter device (DeNOVIX) and trypan blue staining solution.

Substrate functionalization

Substrate functionalization was performed by overnight incubation in phosphate-buffered saline (PBS) with 10 µg/mL CCL21 and 100 µg/mL ICAM1 (produced by the Structural Proteomics Unit, Weizmann Institute, Rehovot, Israel). Detailed information on the protein production and purification can be found in online supplemental information.

Supplemental material

Cell proliferation assay

T cells were stained before seeding with 5 µM carboxyfluorescein succinimidyl ester (CFSE) (BioLegend) for 20 min at 37°C according to the manufacturer’s protocol. Excess dye was removed by washing with five volumes of RPMI. T cells were activated with either anti-CD3/CD28-coated microbeads or antigen-loaded DCs with or without substrate coatings as described above. Three days later, the cells were gently detached from the substrate via a 10 min incubation with PBS without calcium or magnesium, and the pipetting was repeated. Propidium iodide (1 µg/mL; Sigma Aldrich, St. Louis, Missouri, USA) was added to each well for cell death staining. Single-cell suspensions were analyzed on a Bio-Rad ZE5 cell analyzer flow cytometer.

In vitro cytotoxic T-cell killing assay

Target B16 cells expressing OVA and GFP (kindly provided by Guy Shakhar, Weizmann Institute of Science) were suspended in complete medium. Cells were seeded in a 96-well plate (20×103 cells per well) and incubated for 2 hours to enable attachment to the substrate. CD8+ T cells were activated and cultured for 3–7 days with or without substrate coatings as described above and were subsequently added on top of the B16 cells at an effector/target ratio of 3:1 for 48 hours. Time-lapse oblique illumination and florescence images were acquired using a Celldiscoverer 7 microscope (Carl Zeiss) equipped with a Plan-Apochromat 20x/0.95 and a 1x Tubelens connected to an Axiocam 702 camera (Carl Zeiss). Images were taken at 5 min intervals for 48 hours. All the acquired images were analyzed using ImageJ software (https://imagej.nih.gov/ij/).

The extent of cytotoxic activity was quantified by measuring the net cell-associated fluorescence intensity throughout the cytotoxicity assay (0–48 hours) and calculating the residual, normalized fluorescence intensity. The original images were initially filtered to remove uneven backgrounds using the rolling ball algorithm (built in background subtraction in ImageJ) with a diameter of 50 pixels and a sliding paraboloid. Notably, high residual fluorescence scores indicate low cytotoxic activity, and an apparent increase in total fluorescence intensity is attributed to target cell proliferation.

Calculation of “cytotoxicity score”

To quantify the cytotoxic potency of T cells (DC-activated or bead-activated, SIN-treated or untreated), the time at which the target cells-associated fluorescence intensity declined to 50% of the initial level was determined (based on video recording throughout the cytotoxicity assay (0–48 hours)). A “cytotoxic score” corresponding to the rate of elimination of GFP-fluorescent target cells was defined and calculated by dividing the assay duration time (48 hours) by the 50% decline time point, minus 1: (Embedded Image ).

Spectral flow cytometry

CD8+ T cells were activated and cultured for 3, 4 or 7 days with or without substrate coated SIN as described above. Surface and intracellular marker expression were then evaluated by spectral flow cytometry. Brefeldin A (5 µg/mL) and Monensin (2 µM) (BioLegend) were added to the cultures during the last 4 hours of incubation to allow intracellular cytokine accumulation. At the indicated time points, the cells were detached from the substrate, placed in a U-shaped 96-well plate, and washed with PBS+3% FBS (Biological Industries, “washing buffer”). The supernatant was aspirated, and the cells were surface stained (at room temperature for 30 min) with LIVE/DEAD Fixable Blue dead cell stain (1:1000; Invitrogen, Paisley, UK) and with the following fluorescent monoclonal antibodies from the following sources: CD8a-Spark blue 550, CD25-PE-Fire 640, CD69-PerCP, FasL-APC, PD-1-BV421, LAG-3-BV785, and CD62L-BV570 (all from BioLegend); CD44-APC vio 770 (Miltenyi Biotec); and CD127-Alexa 594 (R&D Systems, Minneapolis, Minnesota, USA). The cells were then washed, fixed and permeabilized (BioLegend) and stained (at room temperature for 30 min) intracellularly with antibodies from the following sources: KI67-BV650, Granzyme B-PB, TNF-α-BV750, IL-2-Alexa700 (all from BioLegend), CD107a-Buv395 (BD Biosciences, Oxford, UK) and Perforin-FITC (eBioscience, Hatfield, UK). After washing, the data were acquired using an Aurora (Cytek, California, USA) spectral flow cytometer, and the data were analyzed using FlowJo software (Ashland, Oregon, USA).

RNA extraction, quantification and quality control

RNA-seq of CD8+ T cells was performed to assess changes in the gene expression profile induced by CCL21+ICAM1 SIN. The cells were activated with either αCD3/αCD28 microbeads or antigen-loaded DCs and cultured for 3, 4 or 7 days with or without substrate coatings. At the indicated time points, the cells were harvested, and total RNA was extracted from each sample using an RNeasy Mini Kit (Qiagen, Valencia, California, USA) according to the manufacturer’s instructions. The RNA concentration was estimated using a Qubit 3 Fluorometer (Thermo Fisher Scientific, USA). RNA quality was assessed using TapeStation (Agilent Technologies 4200, USA), and all samples had an RIN>9. RNA aliquots were stored at −80°C until use.

Sample sequencing and gene expression analysis

RNA-seq libraries were prepared at the Crown Genomics Institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine of the Weizmann Institute. A bulk adaptation of the MARS-Seq protocol37 38 was used to generate RNA-Seq libraries. Briefly, 30 ng of input RNA from each sample was barcoded by reverse transcription and pooled. Following Agencourt AMPure XP bead cleanup (Beckman Coulter, California, USA), the pooled samples were subjected to second-strand synthesis and linearly amplified via T7 in vitro transcription. The resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with Illumina sequences during ligation, RT, and PCR. Libraries were quantified by Qubit and TapeStation as previously described.37 38 Sequencing was carried out on a NovaSeq600 using an SP 100 cycle kit (Illumina).

Gene expression analysis

MARS-Seq data were analyzed using the User-friendly Transcriptomic Analysis Pipeline (UTAP) transcriptome analysis pipeline.39 Reads from samples having at least 5 M raw reads were trimmed to remove adapters and low-quality bases using Cutadapt (–a “A(10)” –a “T(10)” –times 2 –u 3 –u −3 –q 20 –m 25) and mapped to the Mus musculus genome (mm10, Gencode annotation) using STAR V.2.4.2a (using the parameters: –alignEndsType EndToEnd, –outFilterMismatchNoverLmax 0.05, –twopassMode Basic, and –alignSoftClipAtReferenceEnds No.). The pipeline quantifies the 3’ region of annotated genes (the 3’ region contains 1000 bases upstream of the 3’ end and 100 bases downstream). Unique molecular identifier (UMI) counting was performed after marking duplicates (in-house script) using HTSeq-count in union mode. Only reads with unique mapping were considered for further analysis, and genes having a minimum of five reads in at least one sample were considered. Gene expression levels were calculated and normalized using DESeq2 with the parameters betaPrior=True, cooksCutoff=FALSE, and independentFiltering=FALSE. Differentially expressed genes were selected if they had an absolute fold change (log2)≥1, an adjusted p≤0.05 (Benjamini and Hochberg) and baseMean≥5. Enrichment analysis was performed using gene set enrichment analysis (GSEA) as described in www.broadinstitute.org/software/gsea. The genes detected by MARS-seq were preranked according to their fold change (log2) in expression between the coated and uncoated treatment groups on each day (day 4 and day 7). Preranked GSEA was applied using the designed 51 T-cell gene sets published by Latis et al 40 (with manual editing of the signature ‘CTL-cytotoxicity and effectors’). Significant gene sets were selected using a cut-off false discovery rate (FDR) q value ≤0.05. Principal component analysis (PCA) of gene expression was performed using UTAP and log-normalized expression values (vst). Volcano plots were generated with MATLAB.

Results

Differential enhancement of T-cell proliferation and antitumor cytotoxicity by SIN stimulation following different activation modes

As a model for antigen-specific T-cell stimulation, we used CD8+ T cells, which were isolated from the spleens of OT-I mice and expressed an OVA-specific TCR. These cells were activated ex vivo, either by coculturing them with OVA-loaded DCs, which serve as antigen-presenting cells, or by treating them with anti-CD3/CD28-coated microbeads and IL-2. To explore the differential effects of DC-mediated (“antigen specific”) and anti-CD3/CD28 bead-mediated (“non-specific”) activation on the response of cells to CCL21+ICAM1 SIN, we systematically determined both the expansion rates and cytotoxicity toward OVA-expressing cancer cells of T cells exposed to the two modes of activation and further cultivated on SIN-coated or uncoated surfaces.

SIN augments the proliferation of T cells following antigen-specific activation

As shown in figure 1A, the substrate-immobilized CCL21+ICAM1 substantially increased the number of viable CD8+ T cells at all time points following activation with OVA-loaded DCs, compared with cells cultured on an uncoated surface. Four days after the initial stimulation, the expansion of SIN-treated T cells was, on average, 2.8±0.4- fold greater (figure 1B), achieving a 30.5±0.5- fold expansion (relative to the number of cells on day 0) for SIN-treated T cells compared with 11.2±1.6- fold expansion for T cells cultured on uncoated surfaces. Following this time point, the expansion potential of the SIN-treated T cells decreased to 1.9±0.1 and 1.3±0.1- fold on days 6 and 7, respectively (see also online supplemental table 1).

Figure 1

CCL21+ICAM1 SIN stimulates T-cell proliferation and antitumor cytotoxicity following DC-mediated or anti-CD3/CD28 bead-mediated activation. CD8+ T cells were activated by antigen-loaded DCs (A–E) or by activation beads (F–J) and cultured for 3–7 days on uncoated or CCL21+ICAM1 SIN-coated culture dishes. The data are shown as the mean±SEM of three independent experiments. The calculated p values (using standard t-tests) are indicated in the figure. (A, F) Bar graphs illustrating the number of live T cells, as determined by trypan blue exclusion. (B, G) Fold expansion ratio of T cells cultured on the SIN coating compared with those cultured on uncoated surfaces. (C, H) Histogram plots of CFSE staining. (D, J) Bar graphs showing the mean fluorescence intensity (MFI) of CFSE. (E, J) Representative overlays of time-lapse oblique illumination (depicting both T cells and target cells) and fluorescence video imaging (see online supplemental movies 1–16) taken at the end of the 48-hour long cytotoxicity assay. The CD8+ T cells (unstained) used in these experiments were seeded prior to coculture with GFP-ovalbumin-expressing target cells, either on an uncoated substrate or on substrate-immobilized CCL21+ICAM1. Notably, live target cells are colored green. Scale bar: 50 µm. One of four independent experiments with similar results is shown here. DCs, dendritic cells; SIN, synthetic immune niche; CFSE, carboxyfluorescein succinimidyl ester.

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We further evaluated the specific effect of SIN on T-cell proliferation. CFSE staining of cells prior to seeding followed by flow cytometry analysis 3 days later showed that the contribution of CCL21+ICAM1 SIN to the elevated cell yield was due to increased T-cell proliferation (figure 1C,D).

SIN augments the proliferation of T cells following antigen-non-specific activation

CCL21+ICAM1 SIN treatment also affected CD8+ T cells that were activated using anti-CD3/CD28-coated microbeads and IL-2. We found that this nonspecific activation yielded fewer T cells (figure 1F) than did antigen-specific activation with DCs (figure 1A); however, SIN treatment significantly and consistently increased the fold expansion values to 5.4±0.4- fold on day 7 following activation with beads (figure 1G), thus reaching a 23.6±0.8- fold expansion for SIN-treated T cells compared with 4.4±0.4- fold expansion for T cells cultured on uncoated surfaces (see also online supplemental table 1). Notably, the augmentation of T-cell proliferation by the SIN, following activation with beads (figure 1C,D,H and I) was considerably higher than its effect on the proliferation of the DC-activated cells.

The differential effects of SIN treatment on the cytotoxic potency of T cells following antigen-specific and non-specific activation

We next tested the effect of substrate-immobilized CCL21+ICAM1 on the capacity of the expanded cytotoxic T cells to kill B16-OVA-GFP melanoma cells following either DC-activation or bead-activation (for details, see Materials and Methods section). Live-cell microscopy-based monitoring of the killing process on selected “key dates” (namely, days 3, 4, 6 and 7) is presented in online supplemental movies 1–16, and the 48-hour “end points” of the cytotoxicity assays are depicted in figure 1E,J (DC-activation) (bead-activation). Quantification of the killing process throughout the cytotoxic assay was performed by recording the mean fluorescence intensity, as shown in online supplemental figures 1 and 2.

As presented in figure 1E,J, online supplemental file 1a,a’, on day 3, the cells displayed substantial killing capacity, irrespective of the mode of activation, although the apparent onset of killing, indicated by a decrease in target-associated fluorescence, was delayed by ~10 hours and reduction in the cytotoxic score in the SIN-treated cells (online supplemental table 1). Moreover, the activation mode per se, had a profound effect on the killing rate and, consequently cytotoxicity score, indicating that the slowly expanding bead-activated cells display higher killing potency than the faster dividing DC-activated cells (online supplemental file 1a,a’ and online supplemental table 1).

Strikingly, on days 4 and 6, SIN treatment had nearly opposite effects on cells activated by the two routes. Specifically, T cells that were activated by OVA-loaded DCs and incubated on uncoated surfaces lost their apparent cytotoxic activity and even displayed a time-dependent increase in fluorescence intensity, which is attributable to persistent target cell proliferation. SIN treatment restored the killing potency of these T cells on both days 4 and 6 (figure 1E, online supplemental movies 3–6 and online supplemental file 1b,c). Notably, SIN treatment had the highest impact on cell expansion rate (three fold increase) on day four and only doubled the expansion rate on day 6 (figure 1B). On the other hand, the bead-activated cells cultured for four or 6 days on uncoated surfaces displayed considerably lower expansion values (figure 1F) but largely retained their killing capacity (figure 1J, online supplemental movies 11–14 and online supplemental file 1b’ and c’ (black curves)). Exposure of these cells to CCL21-ICAM1 SIN strongly enhanced their expansion (figure 1G) but essentially suppressed their cytotoxicity, leading to an increase in target cell-associated fluorescence (figure 1J, online supplemental movies 11–14 and online supplemental file 1b’,c’ (pink curves)). After longer incubation (on day 7), the DC-activated cells were essentially inactive (figure 1 E, online supplemental movies 7 and 8 and online supplemental figure 1d), and the SIN-treated cells displayed a capacity to block further expansion of target cells (compare the pink curve in online supplemental figure 1d to that of the target cells in the absence of T cells; online supplemental figure 2). On the other hand, the bead-activated cells exhibited a sharp phenotypic transition, namely, regaining cytotoxicity following CCL21+ICAM1 SIN treatment while their killing capacity when cultured on uncoated surfaces was limited (figure 1J, online supplemental movies 15 and 16 and online supplemental figure 1d’, compare pink and black curves). Notably, the cytotoxicity presented here was highly antigen-specific, and B16-GFP cells, which do not express OVA, were not killed by OT-I T-cells, irrespective of the treatment (online supplemental movies 17 and 18).

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Notably, in addition to their differential expansion and cytotoxic response to SIN treatment, DC-activated and bead-activated cells also displayed distinct morphological transitions largely corresponding to the cytotoxic potency of the cells. On day 3, the cells displayed a high projected area and elongated shape with high killing capacity, irrespective of the mode of activation (online supplemental movies 1,2,9 and 10). As of day 4 and on, in the absence of SIN, the DC-activated cells underwent morphological transition, characterized by a decrease in the projected cell area and an increase in cell circularity, which was correlated with suppressed killing capacity (online supplemental movies 3 and 5). In contrast, the SIN-treated cells retained their morphology and displayed greater cytotoxic capacity (online supplemental movies 4 and 6). On the other hand, in the bead-activated cells, the morphological transition of the SIN-treated cells, observed on day 4, revealed considerably lower cytotoxic capacity than that observed on day 3 (online supplemental movies 10 and 12). This transition was reversed on day 7 when the cells regained their killing capacity (online supplemental movie 16).

Considering that effective adoptive immunotherapy requires an optimal combination of both a high cell expansion rate and a high level of intrinsic cytotoxic potency, the data presented above indicate that the most effective SIN stimulation timeline is very different for DC-stimulated and bead-stimulated CD8+ T cells (single stimulation). Specifically, for antigen-specific activation, the “optimal effective time point” at which the effect of SIN treatment on cell expansion was maximal (figure 1B) while promoting cytotoxicity (online supplemental figure 1e) was determined to be day 4. On the other hand, for nonspecific activation, the optimal day was day 7 (figure 1G and online supplemental figure 1e ). To further explore the differential activation mode-specific mechanisms involved in SIN stimulation, we employed different molecular profiling approaches, focusing primarily on the ‘effective days’, namely, day 4 and day 7.

Early stages in differential SIN responsiveness, evident on day 4, following antigen-specific and non-specific activation

As shown above, on day 4, some intriguing differential effects of SIN treatment on cytotoxicity and proliferation were observed. The loss of cytotoxic activity in DC-activated T cells was effectively prevented by SIN while the same treatment strongly suppressed the cytotoxic activity of the bead-activated cells. The effect of SIN on the expansion of DC-activated cells reached a maximal effect on day 4 and then declined, while its growth stimulatory effect on bead-activated cells increased for at least three additional days. To explore the molecular mechanisms underlying the differential effects of CCL21+ICAM1 SIN treatment on cytotoxicity and proliferation, we harvested DCs and bead-activated cells at days 3 and 4 and subjected them to spectral flow cytometry using selected phenotypic markers.

As shown in figure 2A,B, exposure of the cells to CCL21+ICAM1 SIN for 3 or 4 days had no significant effect on cell viability following both antigen-specific and nonspecific activation; however, it had major effects on specific differentiation markers. Analysis of the TNaïve cell and memory T-cell subsets revealed that, compared with the control, substrate-immobilized CCL21+ICAM1 substantially increased the prominence of effector T cells (TE) following antigen-specific and nonspecific activation (figure 2C,D). The prominence of effector memory T cells (TEM) among DC-activated cells was also greater for cells cultured on CCL21+ICAM1 substrates than for cells cultured on an uncoated substrate (figure 2C,D).

Figure 2

CCL21+ICAM1 SIN stimulation is associated with a shift in T-cell differentiation toward effector/effector memory subtypes following antigen-specific and nonspecific activation. Flow cytometry analysis of DC-activated and bead-activated T cells (A–D, respectively) cultured for 3 or 4 days on uncoated or SIN-coated surfaces. The gating strategy is shown in online supplemental figure 3. (A, B) Bar graph illustrating the percentage of viable DC-activated and bead-activated CD8+ T cells (mean±SEM) in three independent experiments. The calculated p values (using standard t-tests) are indicated in the figure. The upper pairs represent the differences between day 3 and day 4 for cells cultured on uncoated surfaces (left) and CCL21+ICAM1-coated SIN (right). (C, D) Distribution of subsets of differentiation markers measured on days 3 and 4. The percentages (mean±SEM) of effector T cells (TE, CD44+/CD62L/CD127), effector memory T cells (TEM, CD44+/CD62L/CD127+), central memory T cells (TCM, CD44+/CD62L+) and naïve T cells (CD44-/CD62L+) in the DC-activated and bead-activated CD8+ T-cell populations are shown. The data shown here are representative of three independent experiments. The calculated p values (using standard t tests) for the proportions of effector, effector memory and central memory CD8+ T-cell subsets among the SIN-treated cells were compared with those among the untreated cells (p<0.001, p<0.001 and p<0.05, respectively). DCs, dendritic cells; SIN, synthetic immune niche.

Next, we tested the differential effects of SIN treatment on the expression of key activation, proliferation, cytotoxicity and exhaustion markers on days 3 and 4 following antigen-specific and nonspecific activation. Surprisingly, despite the marked increase in cell proliferation induced by SIN treatment on day 3 following activation with OVA-loaded DCs (figure 1A,B) or with activation beads (figure 1F,G), SIN-treated and untreated cells did not show significant differences in the expression levels of the differentiation markers tested (figure 3A,B). However, on day 4, compared with T cells cultured on uncoated surfaces, treatment of DC-activated cells with SIN elevated the expression of key activation markers, such as CD25 and CD69, and the proliferation markers, such as IL-2 and Ki67 (figure 3A). Analysis of the effect of SIN treatment on the expression of classical components of the killing machinery revealed significant increases in the expression of Fas-L, granzyme B, CD107a, perforin and TNF-α compared with those in cells cultured on uncoated substrates (figure 3A). Cells further exhibited elevated PD-1 expression and decreased LAG-3 expression following SIN stimulation (figure 3A). These observations could account for the augmented killing capacity of DC-activated T cells induced by SIN.

Figure 3

Exposure of DC-activated and bead-activated CD8+T cells to CCL21+ICAM1 SIN for 3 or 4 days had major effects on specific differentiation markers. (A, B) Fold change in the MFI of activation, proliferation, killing and exhaustion markers in DC-activated (A) and bead-activated (B) CD8+ T cells in the presence or absence of SIN stimulation. The data are presented as the mean fold change±SEM relative to nonactivated cells and were obtained from three independent experiments. The calculated p values (using standard t tests) are indicated in the figure. The upper pairs represent the differences between day 3 and day 4 for cells cultured on uncoated surfaces (left) and CCL21+ICAM1-coated SIN (right). DCs, dendritic cells; MFI, mean fluorescence intensity; SIN, synthetic immune niche.

Examination of the SIN-induced effects on bead-activated cells on day four revealed a very different pattern, manifested by decreased expression of the late activation marker CD25 while still inducing higher expression levels of the early activation marker CD69 (which was also elevated in the SIN-treated DC-activated cells) (figure 3B) and elevated expression of the proliferation markers IL-2 and Ki67 (compared with the control) (figure 3B). Additionally, in contrast to the effects of SIN following DC activation, which led to an increase in the expression levels of key killing markers, similar treatments of bead-activated cells resulted in the suppression of Fas-L, granzyme B and TNF-α expression. The level of CD107a was still increased, but no significant difference was evident in the level of perforin expression (figure 3B). Notably, in contrast to DC activation, SIN treatment decreased the PD-1 expression level on day four and similarly reduced the LAG-3 expression. Taken together, these results provide a broad mechanistic view of the molecular basis underlying the suppression of cytotoxicity (and strong enhancement of proliferation) in bead-activated cells and the enhancement of cytotoxicity (combined with reduced enhancement of proliferation) in DC-activated cells following 4 days of treatment with SIN.

To further obtain a broad and unbiased assessment of the differential molecular responses to SIN treatment following antigen-specific and nonspecific activation, we conducted RNAseq analyses at specific “effective timepoints”, namely, day 4 (for the DC-activated cells) and day 7 (for the bead-activated cells).

RNAseq-based unbiased molecular phenotyping of SIN-stimulated CD8+ T cells 4 days after antigen-specific stimulation

Differential transcriptomic profiling of SIN-treated and untreated T cells was performed on days 3 and 4 following antigen activation (online supplemental table 2). PCA of the profiling data showed that the “control group” (day 0—non-activated samples) was clearly separated (with PC1) from the bead/DC-activated samples and that the day 3 samples could be distinguished (with PC2) from the days 4 and 7 samples (online supplemental figure 4). The genes differentially expressed between SIN-treated cells and untreated cells that exhibited changes in DC-activated cells on day three were strongly correlated with the changes in bead-activated cells (correlation coefficient (r)=0.6109), revealing that most of the changes were not specific to the type of activation (online supplemental figure 5a,b). Unbiased RNAseq analysis on day four following antigen activation revealed significant changes in the differential expression of 368 genes (online supplemental table 3) and (figure 4A). Among them, we detected relevant signatures that were significantly enriched in SIN-treated CD8+ T cells, namely, “proliferation”, “CTL cytotoxicity” and the “memory up” signature40 (figure 4A), which included genes upregulated in memory T cells. Closer inspection of individual genes within these enriched signatures revealed that consistent with the flow cytometry data, SIN-treated CD8+ T cells exhibited a gene expression profile clearly skewed toward the effector phenotype, with upregulation of genes associated with T-cell activation, such as Il2ra and Pdcd1, and genes involved in T-cell cytotoxicity, such as Gzma, Gzmb, Gzmc, Ifng and Tnf (figure 4B). We also observed the upregulation of genes involved in T-cell migration, such as the chemokine receptors Cx3cr1, Ccr2 and Ccr5, as well as genes encoding costimulatory receptors (Havcr2) (figure 4B). Importantly, in DC-activated/SIN-stimulated cells, the expression of genes encoding inhibitory receptors such as Lag3 and Klrd1 was downregulated, as was the expression of genes encoding molecules mediating immunosuppressive signals (Il10ra) (figure 4C).

Supplemental material

Supplemental material

Figure 4

Transcriptional profiling of SIN-stimulated CD8+ T cells harvested on day four after activation by antigen-loaded DCs. (A) Volcano plot of gene expression following SIN stimulation showing the upregulation of genes involved in T-cell activation and cytotoxicity (shown on the right) and the downregulation of genes associated with exhaustion (on the left). The genes are colored according to their assigned functional signatures. Dashed lines mark the cut-off differentially expressed genes (absolute fold change (log2)≥1 and adjusted p≤0.05). (B) Normalized gene expression values detected by DESeq2 analysis for selected upregulated genes or (C) selected downregulated genes. The horizontal lines indicate the medians. DCs, dendritic cells; SIN, synthetic immune niche.

RNAseq-based unbiased molecular phenotyping of SIN-stimulated CD8+ T cells 7 days after antigen non-specific stimulation

To further characterize the transcriptomic alterations induced by SIN treatment, we activated CD8+ T cells with beads for 7 days (the ‘optimal effective day’ for bead-activated cells) with or without CCL21+ICAM1 SIN coating and analyzed their cellular and gene expression profiles.

As shown in figure 5A, after 7 days of culture, 75% of the cells stimulated with CCL21+ICAM1 SIN were still alive, whereas only 36% of the nontreated cells remained, indicating that this SIN significantly promoted cell survival following bead activation. Our data also revealed that following such activation, the CCL21+ICAM1 coating significantly increased the prominence of effector T cells (TE) compared with that in cells growing on uncoated substrates (figure 5B). This effect was largely similar to that induced in DC-activated cells (figure 2C) but had only a marginal effect on the prominence of effector memory (TEM) T cells (figure 5B). These SIN-treated T cells were further characterized for the expression of specific activation, proliferation, killing and exhaustion markers (CD25, CD69, IL-2, Ki67, Fas-L, granzyme B, CD107a, perforin, TNF-α, PD-1 and LAG3). As shown in figure 5C, SIN-treated T cells displayed increased expression of IL-2 compared with that in T cells cultured on uncoated surfaces, whereas no significant difference was evident in the expression levels of CD25, granzyme B, perforin or TNF-α. Moreover, the cellular levels of CD69, Ki67, Fas-L and CD107a were significantly lower in cells exposed to the coated surfaces than in those cultured on uncoated surfaces. The cells were further characterized for the expression of the inhibitory molecules PD-1 and LAG-3. As shown, compared with T cells cultured on uncoated surfaces, SIN-treated T cells displayed increased expression of LAG-3, whereas no significant difference was evident in the expression of PD-1 (figure 5C). Taken together, while the SIN-treated cells displayed an enhanced killing capacity on day 7, the differential expression levels of cytotoxicity-related markers between the SIN-treated and untreated cells did not indicate it.

Figure 5

Prolonged stimulation of bead-activated T cells with CCL21+ICAM1 SIN increases T-cell survival and alters the prominence of subsets of differentiation markers. Flow cytometry staining was performed for T cells that were bead-activated and further cultured for 7 days with or without CCL21+ICAM1 SIN. The gating strategy is shown in online supplemental figure 3. (A) Bar graph illustrating the percentage of viable CD8+ T cells as determined by flow cytometry analysis. The data are shown as the mean±SEM of three independent experiments. The calculated p values (using standard t tests) are indicated in the figure. (B) Distribution of differentiation subset markers in these cells on day 7. The percentages (mean±SEM) of effector T cells (TE, CD44+/CD62L/CD127), effector memory T cells (TEM, CD44+/CD62L/CD127+), central memory T cells (TCM, CD44+/CD62L+) and naïveT cells (CD44-/CD62L+) among the CD8+ T cells are shown. The data shown here are representative of three independent experiments. The calculated p values (using standard t-tests) for the comparisons of the proportions of effector, effector memory and central memory CD8+ T-cell subsets among the SIN-treated cells and the untreated cells were calculated (p<0.01, ns and p<0.001, respectively). (C) Fold change in the MFI of activation, proliferation, killing and exhaustion markers in bead-activated CD8+ T cells in the presence or absence of SIN stimulation, measured on day 7. The data are presented as the mean fold change±SEM relative to nonactivated cells and were obtained from three independent experiments. The calculated p values (using standard t-tests) are indicated in the figure. The pairs of bars represent the MFI of cells cultured on uncoated (left) and SIN-coated (right) surfaces. MFI, mean fluorescence intensity; SIN, synthetic immune niche.

The gene expression profiles of T cells revealed differential expression of 302 genes in SIN-treated CD8+ T cells compared with T cells cultured on uncoated surfaces (online supplemental table 4 and figure 6A). Specifically, the “pro-apoptotic”, “exhaustion” and “CTL cytotoxicity” signatures were significantly enriched in untreated CD8+ T cells. A deep analysis of specific genes within enriched signatures revealed the downregulation of proapoptotic genes, such as Bmf, Bbc3, Bcl2l11 and Fas, as well as genes associated with exhausted cells, such as Lag3, Ctla4, Havcr2, Mx1, Egr2 and klrd1 (figure 6B). Importantly, compared with those in the controls, the expression of genes involved in T-cell cytotoxicity (Gzmm) was upregulated (figure 6C).

Supplemental material

Figure 6

Transcriptional profiling of SIN-stimulated CD8+ T cells harvested on day seven after activation with anti-CD3/CD28-coated microbeads. (A) Volcano plot of gene expression following SIN stimulation showing the upregulation of genes involved in T-cell cytotoxicity (shown on the right) and the downregulation of proapoptotic and exhaustion-related genes (on the left). The genes are colored according to their assigned functional signatures. Dashed lines mark the cut-off differentially expressed genes (absolute fold change (log2) ≥1 and adjusted p≤0.05). (B) Normalized gene expression values detected by DESeq2 analysis for selected downregulated genes or (C) selected upregulated genes. The horizontal lines indicate the medians. SIN, synthetic immune niche.

To gain insight into the differential regulation of cell proliferation and cytotoxicity in SIN-treated cells following DC or bead activation, we compared the transcriptomic profiles of the respective CD8+ T cells on their “optimal effective days” (day 4 and day 7). As shown in figure 7, a comparison of the changes in DC or bead activation revealed a decreased correlation (correlation coefficient (r)=0.2063). Specifically, SIN-treated T cells activated with DCs had significantly greater expression levels of genes related to T-cell activation, cytotoxicity and migration than bead-activated cells. In contrast, we noticed decreased levels of proapoptotic genes and genes associated with exhausted cells, mostly following bead activation, suggesting that SIN stimulation may induce decreased expression of genes involved in the regulation and dampening of immune responses. In addition, similar to those in bead-activated T cells, the expression of the inhibitory molecules Lag3 and Klrd1 was significantly downregulated in DC-activated T cells following SIN stimulation. However, we found that the costimulatory/inhibitory molecule Havcr2 is expressed at higher levels in DC-activated cells than in bead-activated cells.

Figure 7

Comparison of the significant differences in gene expression profiles between DC-activated cells (orange dots) and bead-activated cells (black dots) on their ‘optimal effective days’ (days 4 and 7, respectively). The orange circle indicates selected genes associated with T-cell activation, cytotoxicity and migration; these genes were elevated in the DC-activated cells on day 4. The black circle indicates genes associated with pro-apoptosis and exhaustion that were downregulated in the bead-activated cells on day 7. DC, dendritic cell.

Discussion

A major challenge encountered in adoptive cancer immunotherapy is the inherent process of exhaustion of CD8+ T cells, which suppresses their proliferation, cytotoxic potency or both.41–43 Recent studies indicate that the exhaustion transition is a complex, multistage process that is driven by different features of the tumor microenvironment,18 such as prolonged antigen presentation, exposure to suppressive cytokines and tumor-derived metabolites.44 Notably, investigations carried out in cancer patients, diverse animal models and ex vivo settings have shown that the exhaustion process is heterogeneous at both the molecular and functional levels45–47 and that its differential effects on T-cell proliferation and killing capacity are still poorly understood.

This notion motivated us to develop an artificial environmental system termed the SIN for the reinforcement of adoptive cancer immunotherapy by optimizing the balance between T-cell expansion and functionality. In these studies, we showed that costimulation of activated murine CD4+35 and CD8+ 15 T cells with SIN, which consists of immobilized CCL21 and ICAM1, enhances the proliferation of both cell types and further augments the cytotoxic activity of CD8+ cells both in culture and in vivo.15 We further showed that SIN also supports the proliferation and functionality of human TILs.35 The general features of the effects of SIN on T cells have been well established; however, the molecular mechanisms underlying these effects of CCL21-ICAM1 SIN have not been elucidated.

In the present study, we tested the day-by-day effects of SIN stimulation on the fine interplay between T-cell proliferation and cytotoxic potency and conducted detailed temporal profiling of the expression of relevant molecular regulators and markers that are associated with T-cell activation, proliferation, subset differentiation, cytotoxicity and exhaustion. Furthermore, given that the effects of SIN were shown to require T-cell activation,48 we further explored the differential effects of SIN following T-cell activation induced by antigen-dependent DC-mediated treatment or antigen-independent activation via bead-conjugated anti-CD3/CD28 antibodies.

The results presented in figure 1 and the related online supplemental movies and the temporal quantification of the killing process (online supplemental figure 1) clearly reveal some major differences in the interplay between expansion (figure 1A,F) and cytotoxic potency (figure 1E,J) in T cells activated by DCs or beads in the presence or absence of the CCL21-ICAM1 SIN. Briefly, in the absence of SIN treatment, DC-activated cells exhibited continuous proliferation, achieving an 11.2-fold increase on day four relative to the number of cells on day 0. SIN treatment enhanced this expansion rate, reaching maximal levels on day 4 (threefold increase over cells cultured on the uncoated surface). After longer incubation, the effect of SIN gradually decreased. The bead-activated cells displayed a poor expansion rate, reaching only 4.4-fold expansion on day seven relative to the number of cells on day 0, and this value was dramatically enhanced on SIN stimulation in a time-dependent fashion (reaching a further 5.4-fold enhancement on day seven relative to cells cultured on the uncoated surface. Daily testing of the cytotoxic phenotypes of the cells indicated that up to and including day 3, the cells displayed high and largely comparable cytotoxic potency, irrespective of the mode of activation or SIN treatment. In contrast, after 4 days, cell fate was strongly and differentially affected by both the mode of activation and the SIN treatment. Specifically, the cytotoxic potency of DC/OVA-activated cells was strongly reduced on day four but could be effectively rescued by SIN treatment while bead-activated cells retained their full cytotoxic properties up to day 6; however, surprisingly, this activity was strongly suppressed by SIN on days 4 and 6. Interestingly, after further incubation of the SIN-treated cells for one additional day (day 7) when the cells reached their maximal SIN-induced expansion rate (5.4-fold, compared with the ‘uncoated’ control), the cytotoxic activity was restored, suggesting that the high cell proliferation rate in these cells is compatible with the recovery of high T-cell killing functionality, provided that the cells were treated with SIN.

These results clearly indicate that in the absence of SIN, the cytotoxic potency of the T cells (as revealed by end-point assessment of cytotoxicity (figure 1E,J) or killing dynamics quantification (online supplemental figure 1 and online supplemental table 1) is inversely correlated with their respective proliferation rates (figure 1A,F, and online supplemental table 1). Thus, the killing capacity of the proliferative DC-activated cells was rather limited and was apparently suppressed on day 4 while the slow-proliferating bead-activated cells retained their full killing activity through day 7. This notion is also in line with recent in vivo studies showing that highly proliferative dysfunctional CD8+ TILs are found in patients with melanoma49 and non small cell lung cancer (NSCLC).50 51 Most importantly, our data indicate that SIN treatment can modulate this suppressive effect, for example, by maintaining cytotoxic potency on day 4 following DC activation and on day 7 in bead-activated cells while strongly enhancing proliferation. These results indicate, in fact, that T cells activated by OVA-loaded DCs or bead-conjugated anti-CD3/CD28 antibodies reach their maximal “beneficial” SIN effects (manifested by retention of high killing potency and high overall cell yields) at very different time points, namely, on day 4 and day 7, respectively (see online supplemental table 1).

Notably, some of the functional changes in cell potency were rather rapid (occurring within 1 day), as was the loss of cytotoxic potency on day four in DC-activated cells without SIN, the loss of killing capacity by bead-activated and SIN-treated cells on the same day and the recovery of cytotoxic activity during the transition of these cells on day 7. Other effects, such as the retention of cytotoxic agents in the DC-activated cells on days 4 and 6, which decreased only on day 7, persisted for longer durations.

Molecular profiling, based on spectral flow cytometry and RNAseq analyses, was used to characterize the possible molecular mechanisms underlying the functional transitions revealed by the “phenotypic profiling” described above, focusing mainly on the following three questions: (1) What is the molecular mechanism responsible for the loss of cytotoxic activity in DC-activated cells and its restoration by SIN treatment? (2). What is the basis for the SIN-induced arrest of cytotoxicity in bead-activated cells on day 4? (3) How similar are the molecular setups of the DC-activated and bead-activated and SIN-stimulated cells on their “optimal effective days”, namely, day 4 and day 7?

We found that on day 3 following activation with OVA-loaded DCs or activation beads, SIN-treated and untreated cells did not exhibit significant differences in the expression levels of markers associated with T-cell cytotoxicity (figure 3A,B). Concerning the DC/OVA-activated cells on day 4, in the absence of SIN, the cells displayed decreased levels of key killing markers, such as Fas-L, granzyme B, CD107a and perforin, compared with their levels on day 3 (figure 3A). SIN treatment clearly blocked the decreases in the levels of these markers (figure 3A), suggesting that the downregulation of these cytotoxicity drivers is responsible for the reduced cytotoxicity. Another interesting transition of the DC-activated cells was the SIN-dependent increase in the expression of activation (CD25 and CD69) and proliferation (IL-2 and Ki67) markers (figure 3A). As activation and proliferation markers are indicators of cytotoxicity,52 their increased expression in SIN-treated cells might support the persistence of cytotoxic potency on day 4. The functional significance of CCL21+ICAM1 stimulation of DC-activated cells was also manifested by a shift in the T-cell differentiation pathway toward effector/effector memory subtypes (figure 2C). Adoptive cell therapy involving the combination of CD8+ T cells was previously shown to have superior efficacy over traditional adoptive therapy involving effector CD8+ T cells alone, leading to greater suppression of melanoma tumor growth, potentially due to complementary cell killing patterns and local production of IL-2 by memory cells.53

To further validate our flow cytometry results, in an unbiased fashion, we studied the differentially expressed genes in DC-activated cells that were affected by SIN treatment. Consistent with our flow cytometry data, we found that SIN-treated cells acquired an effector phenotype manifested by the upregulation of genes associated with T-cell activation, cytotoxicity and migration (figure 4A,B). Notably, we also detected increased expression of the exhaustion marker PD-1 in SIN-treated cells (figure 4A,B), which is often exploited by tumors for immune evasion and may impair T-cell-mediated killing on ligation to its ligand PD-L1.54 55 Nonetheless, PD-1 expression on antigen-specific T cells reflects the avidity and antitumor reactivity of these T cells.56–58 Thus, PD-1 expression may be independent of T-cell exhaustion and can be considered a marker of activated tumor-reactive T cells. Notably, we found that PD-1-expressing DC/OVA-activated cells exhibited a clearly multifunctional profile and decreased PD-L1 expression (Cd274, figure 4A,C), indicating that PD-1 expression does not necessarily reflect dysfunction or exhaustion. Finally, we observed upregulation of the Havcr2 gene (figure 4A,B), which encodes TIM-3. This observation may be of particular interest because previous studies have shown that TIM3 functions as a coinhibitory receptor,59 but it was also suggested that TIM3 might act as a costimulatory receptor.60 61 Our results showed that SIN-treated cells exhibit high functional capacity, suggesting that Tim-3 is more similar to costimulatory receptors that are upregulated following T-cell activation than to a dominant inhibitory protein such as Lag3 or Ctla4.

The molecular mechanism of the bead-activated cells on days 3 and 4 was very different. As indicated above, in the absence of SIN, the expansion of the cells was 2.0-fold and 3.1-fold slower than that of their DC-activated counterparts, and consequently, the effect of SIN was very high (4-fold and 3.5-fold, respectively). This is consistent with the increase in the levels of CD69, IL-2 and Ki67.

In contrast to the effects of SIN on DC-activated cells, on day 4, similar treatment with bead-activated T cells suppressed the expression of Fas-L, granzyme B and TNF-α. Interestingly, at the same time, the levels of the exhaustion markers PD-1 and LAG-3 also decreased (figure 3B), suggesting that these molecules attenuated the proliferation of the bead-activated cells and that their suppression (possibly by SIN stimulation) was responsible for the remarkable increase in the proliferation rate (see also references 62 63 and discussion in figure 6 below). Notably, while the reduction in the Fas-L level was substantial, the residual levels of granzyme B and TNF-α were quite high and comparable to their levels on day 3, suggesting that either the cytotoxicity of these cells was primarily Fas-L dependent or that it was downregulated by SIN downstream of granzyme B and TNF-α. Given these considerations, it could be proposed that SIN-induced proliferation (directly or by suppressing exhaustion) is involved in the arrest of killing potency.

To compare the molecular profiles of the DC-activated and bead-activated cells on their optimal effective days (day 4 and day 7, respectively), we conducted flow cytometry analysis of relevant functional markers (activation, proliferation, killing and exhaustion) and differentiation subtyping, as well as differential transcriptional analysis of the bead-activated cells on day 7 (figures 5 and 6). These results were subsequently compared with the corresponding results of the effects of SIN on DC-activated cells on day 4, as shown in figures 2A,B, 3A and 4.

Unlike the effects of SIN following DC activation, on day 4, similar treatment with the bead-activated cells for 7 days significantly promoted cell survival (compare figures 2A and 5A). Furthermore, we found that SIN treatment significantly increased the percentage of effector T cells (figure 5B), similar to what was observed in DC-activated cells (figure 2C) but had only a marginal effect on the percentage of effector memory T cells (TEM) (figure 5B). Effector CD8+ T cells are preferred for adoptive T-cell therapy because of their rapid proliferative capacity and ability to engage and clear tumor cells,53 suggesting that CCL21+ICAM1 may be used in the ex vivo processing of T cells toward adoptive cell therapy. Additionally, in contrast to the effects of SIN on DC-activated cells, which led to an increase in the expression levels of key activation, proliferation and killing markers (figure 3A), similar treatment of bead-activated cells resulted in the suppression of CD69, Ki67, Fas-L and CD107a expression (figure 5C). In addition, similar to that in DC-activated T cells, the expression of the inhibitory molecule Lag3 was significantly reduced in bead-activated T cells following SIN stimulation (figures 3A and 5C).

Consistent with our flow cytometry data, we observed lower expression levels of genes encoding inhibitory receptors (Lag3, Ctla4 and Havcr2; figure 6A,B). Decreased expression of genes involved in T-cell regulation may be associated with the regained T-cell function observed on day seven in SIN-treated cells, as blocking LAG-3,64 CTLA-465 or Tim-366 was previously reported to be effective at restoring antitumor immunity and promoting tumor regression. In addition, Gzmm was expressed at higher levels in SIN-treated cells than in control cells (figure 6C). Gzmm has been shown to efficiently induce cell death in tumor cells and inhibit cytomegalovirus replication in a noncytotoxic manner.67

To increase the power of our analysis, we combined the gene profiles of DC-activated and bead-activated CD8+ T cells (figure 7). Notably, in DC-activated cells, the primary contribution of SIN is the upregulation of activation-associated, cytotoxicity-associated and migration-associated genes, while in bead-activated cells, it is the suppression of proapoptotic and exhaustion-associated genes.

Conclusion

We demonstrated here the capacity of a CCL21+ICAM1-based SIN to induce an “optimal interplay” between the expansion and cytotoxicity of CD8+ T cells. Specifically, we showed that integrated analysis of specific differentiation markers by flow cytometry, together with gene expression profiling, contribute to the elucidation of the molecular mechanism underlying SIN stimulation. Based on these results, we propose that the CCL21+ICAM1 SIN can be used for increasing the efficacy of the ex vivo expansion of T cells for adoptive immunotherapy.

Supplemental material

Data availability statement

Data are available in a public, open access repository. The datasets supporting the conclusions of this article are included within the article and have been deposited in NCBI’s Gene Expression Omnibus (GEO) and is available through GEO series accession number GSE254335 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE254335). For further information on original data, contact the corresponding author.

Ethics statements

Patient consent for publication

Ethics approval

Experiments were performed under protocols approved by the Weizmann Institute's Institutional Animal Care and Use Committee (https://www.weizmann.ac.il/pages/animal-research, Protocol number: 02020220-3).

Acknowledgments

We would like to express our gratitude to Tamar Unger and Shira Albeck of the Dana and Yossie Hollander Center for Structural Proteomics (WIS) for the expert production of the SIN components used in this project. We would like to thank Yoseph Addadi and Inna Goliand from the de Picciotto Cancer Cell Observatory (WIS), and Ekaterina Kopitman and the Flow Cytometry Unit (WIS) for their expert technical assistance.

References

Supplementary materials

Footnotes

  • Deceased NF is deceased

  • Contributors Conceptual and experimental design: SY, RZ, SR-Z, NF and BG. Performance of the experiments: SY and RZ. Data analysis and quantification: BD, SY, RZ, SR-Z and BG. Manuscript writing and approval: SY, BD, RZ, SR-Z and BG. Author acting as guarantor: BG.

  • Funding The experiments described here were supported by the Israel Science Foundation (IPMP; #3617/19), the Volkswagen Foundation grant # 7136510302, and internal grants from the Weizmann Institute of Science (WIS; Brunschwig Jean Marc support).

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.